Metabolites Profile of Extracts and Fractions of Erythroxylum mexicanum Kunth by UHPLC-QTOF-MS/MS and its Antibacterial, Cytotoxic and Nitric Oxide Inhibitory Activities.

The present study shows the untargeted metabolite profiling and in vitro antibacterial, cytotoxic, and nitric oxide (NO) inhibitory activities of the methanolic leaves extract (MLE) and methanolic stem extract (MSE) of Erythroxylum mexicanum, as well as the fractions from MSE. Using ultra-high performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS), a total of 70 metabolites were identified; mainly alkaloids in the MLE, while the MSE showed a high abundance of diterpenoids. The MSE fractions exhibited differential activity against Gram-positive bacteria. Notably, the hexane fraction (HSF) against Streptococcus pyogenes ATCC 19615 (MIC=62.5 µg/mL) exhibited a bactericidal effect. The MSE fractions exhibited cytotoxicity against all cancer cell lines tested, with selectivity towards them compared to a noncancerous cell line. Particularly, the HSF and chloroform fraction (CSF) showed the highest cytotoxicity against prostate cancer (PC-3) cells, with IC50 values of 19.9 and 18.1 µg/mL and selectivity indexes of 3.8 and 4.2, respectively. Both the HSF and ethyl acetate (EASF) fractions of the MSE inhibited NO production in RAW 264.7 macrophages, with NO production percentages of 50.0 % and 51.7 %, respectively, at a concentration of 30 µg/mL. These results indicated that E. mexicanum can be a source of antibacterial, cytotoxic, and anti-inflammatory metabolites.

Cytotoxic Activity of Wild Plant and Callus Extracts of Ageratina pichinchensis and 2,3-Dihydrobenzofuran Isolated from a Callus Culture.

Ageratina pichinchensis (Kunth) R.M. King & H. Rob. belongs to the Asteraceae family and is a plant native to Mexico to which several biological properties are attributed. In this study, the cytotoxic effect of four extracts from the wild plants and two extracts from A. pichinchensis callus culture were evaluated against carcinogenic cell lines including prostate carcinoma, cervical cancer, hepatocellular carcinoma, hepatoma human, lung cancer, and cellular keratinocytes. The extracts were obtained with ethyl acetate and methanol using both leaves and stems or the callus. Only the ethyl acetate extract of the callus culture influenced the cervical cancer cell line (HeLa) with an IC50 of 94.79 ± 2.0 µg/mL. From the ethyl acetate callus extract, 2,3-dihydrobenzofuran was isolated and purified and also evaluated against cancer cells. The cytotoxic evaluation of this compound showed a significant effect against the HeLa cell line with an IC50 of 23.86 ± 2.5 µg/mL. Our results contribute to the development of biotechnological alternatives and extraction processes to produce compounds with possible potential against certain types of human cancer.

Obtaining 2,3-Dihydrobenzofuran and 3-Epilupeol from Ageratina pichinchensis (Kunth) R.King & Ho.Rob. Cell Cultures Grown in Shake Flasks under Photoperiod and Darkness, and Its Scale-Up to an Airlift Bioreactor for Enhanced Production.

Ageratina pichinchensis (Kunth) R.King & Ho.Rob. is a plant used in traditional Mexican medicine, and some biotechnological studies have shown that its calluses and cell suspension cultures can produce important anti-inflammatory compounds. In this study, we established a cell culture of A. pichinchensis in a 2 L airlift bioreactor and evaluated the production of the anti-inflammatory compounds 2,3-dihydrobenzofuran (1) and 3-epilupeol (2). The maximum biomass production (11.90 ± 2.48 g/L) was reached at 11 days of culture and cell viability was between 80% and 90%. Among kinetic parameters, the specific growth rate (µ) was 0.2216 days-1 and doubling time (td) was 3.13 days. Gas chromatography coupled with mass spectrometry (GC-MS) analysis of extracts showed the maximum production of compound 1 (903.02 ± 41.06 µg/g extract) and compound 2 (561.63 ± 10.63 µg/g extract) at 7 and 14 days, respectively. This study stands out for the significant production of 2,3-dihydrobenzofuran and 3-epilupeol and by the significant reduction in production time compared to callus and cell suspension cultures, previously reported. To date, these compounds have not been found in the wild plant, i.e., its production has only been reported in cell cultures of A. pichinchensis. Therefore, plant cell cultured in an airlift reactor can be an alternative for the improved production of these anti-inflammatory compounds.

Arnica montana Cell Culture Establishment, and Assessment of Its Cytotoxic, Antibacterial, α-Amylase Inhibitor, and Antioxidant In Vitro Bioactivities.

Arnica montana cell suspension culture could be a sustainable source of a vegetal material producer of secondary metabolites (SMs) possessing biological effects. Different plant growth regulator concentrations (0-5 mg/L) were tested in foliar explants to induce a callus that was used to establish a cell suspension culture. Growth kinetics was carried out for 30 days. A methanolic extract obtained from biomass harvested at 30 days of growth kinetics was fractionated, and three fractions were tested for bioactivities. We induced a callus with 1 mg/L of picloram and 0.5 mg/L of kinetin in foliar explants, which allowed for the establishment of a cell suspension culture, and the latter had the highest total SMs contents at day 30. Three fractions showed differences in total SMs contents, with the highest values per gram as follows: 270 mg gallic acid equivalent for total phenolic content, 200 mg quercetin equivalent for total flavonoid content, 83 mg verbascoside equivalent for total phenolic acid content, and 396 mg parthenolide equivalent for total sesquiterpene lactone content. The best bioactivities were 2-6 µg/mL for the 50% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical, 30% cellular viability of lymphoma cells at 40 µg/mL, 17% inhibition against Escherichia coli and Staphylococcus aureus at 8 µg/disk, and α-amylase inhibition at 12% with 10 µg/mL. The total SMs contents were correlated with bioactivities.

In vivo anti-arthritic effect and repeated dose toxicity of standardized methanolic extracts of Buddleja cordata Kunth (Scrophulariaceae) wild plant leaves and cell culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Buddleja cordata Humb. Bonpl. & Kunth, known by the population as Tepozán blanco, is a shrub plant used in traditional herbal medicine in Mexico for the treatment of tumors, cancer, sores, skin burns, rheumatic pains and diseases related to inflammatory processes such as arthritis; authors adjudicate this etno-medicinal effect to the presence of secondary metabolites in the plant such as verbascoside, however due to its low concentration in recent years biotechnological tools are applied as cell culture to biosynthesize these pharmacological active metabolites in greater quantities. AIM OF THE STUDY: Evaluate the possible toxic effect after a daily administration of MeOH extracts from wild plant leaves (Bc-Wp), and cell culture (Bc-Cc) of B. cordata for 28 days, and after their anti-edematous and antioxidant activities in vivo, as well their effect on the cytokines profile during experimental arthritis induced by complete Freund's adjuvant. MATERIALS AND METHODS: Both extracts were evaluated in CD1 male mice first in a toxicity test of repeated dose administrations (1 g/kg) for 28 days, after which pharmacological activity of both extracts was measure during experimental induced arthritis where three doses were tested, at the end of the study edema formation, body weight gain and antioxidant activity were measure in edema and ganglionic tissues. Finally, dose that exerted the best protective effect (250 mg/kg) was evaluated to quantify its effect over ganglionic tissue concentration of lymphocytes T CD4+, and cytokines (IL-1ß, TNF-α and IL-10), as well histological analysis in arthritic mice. RESULTS: Both extracts (Bc-Wp and Bc-Cc) did not exert lethality, however body weight gain and food in-take were lower than in healthy mice administered with vehicles, also extract-treated animals showed a decrease in serum lipid concentration and only Bc-Wp extract treated animals decrease serum alkaline phosphatase after 28 daily administration compared to healthy un-treated mice. During experimental arthritis best inhibition effect over edema development was observed in those animals administered with both extracts at dose of 250 mg/kg (Bc-Wp and Bc-Cc) on day 28, compared to CFA un-treated mice. Also both extracts reduce oxidative damage over lipids and proteins at the same dose, in both ganglionic and edema tissue, as well antioxidant enzymatic response was reduced in both tissues compared to arthritic un-treated group. In ganglionic tissue of arthritic mice, CD4+ lymphocytes concentration was reduced by Bc-Wp and Bc-Cc treatment (250 mg/kg) respectively, as well IL-1ß, and TNF-α levels. Only arthritic animals treated with Bc-Cc extract at 250 mg/kg generated a significant increase of IL-10 doubling the levels compared to CFA un-treated group. Histological analysis of popliteal ganglion showed that both extracts decrease the incidence of lytic lesions, lipid inclusions and leukocyte infiltration. CONCLUSION: Methanolic extracts of wild Buddleja cordata and its cell cultures did not generated lethality after a daily administration for 28 days at 1 g/kg, but it was observed that both showed a lipid-lowering effect. Also at dose of 250 mg/kg both extracts exerted anti-edematous, protection against the oxidation of lipid and proteins, regulation on antioxidant enzymatic response, down-regulation on lymphocytes CD4+ producers of IL-1ß and TNF-α, an increase in IL-10 levels, which caused a decrease in leukocyte infiltration in ganglionic tissue during experimental arthritis.

Establishment and Phytochemical Analysis of a Callus Culture from Ageratina pichinchensis (Asteraceae) and Its Anti-Inflammatory Activity.

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L-1 NAA with 0.1 mg L-1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 µM), IL-6 (IC50 = 73.71 ± 3.21 µM), and TNF-α (IC50 = 73.20 ± 5.99 µM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 µM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.

Production of dihydroxylated betalains and dopamine in cell suspension cultures of Celosia argentea var. plumosa.

Betalains are plant pigments of hydrophilic nature with demonstrated chemopreventive potential in cancer cell lines and animal models. Among the betalains, those containing an aromatic moiety with two free hydroxyl groups possess the strongest antioxidant and free radical scavenging activities. The betaxanthins dopaxanthin and miraxanthin V and the betacyanins betanidin and decarboxy-betanidin are the only natural betalains with catecholic substructures. These four pigments have been produced in cell cultures established from hypocotyls of the plant Celosia argentea. Two stable and differentially colored cell lines, yellow and red, were maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (6.66 µM) and 2,4-dichlorophenoxyacetic acid (6.79 µM). Derived suspension cultures showed increased production of dihydroxylated betalains in the cells and secreted to the medium with a maximum reached after 8 days of culture. In addition, precursor molecules betalamic acid and dopamine, with content up to 42.08 mg/g dry weight, were also obtained. The joint presence of the bioactive betalains together with the production of dopamine and betalamic acid show the ability of cell cultures of C. argentea to become a stable source of valuable phytochemicals.

Honokiol and magnolol production by in vitro micropropagated plants of Magnolia dealbata, an endangered endemic Mexican species.

An efficient protocol for the in vitro propagation of Magnolia dealbata Zucc., an important medicinal plant that is the source of the anxiolytic and anticancer compounds honokiol and magnolol, was established. This plant is wild-crafted, and conservationists have expressed concerns with regard to the sustainability of production. In the present work, two factors were found to be of importance for the regeneration of M. dealbata and the production of honokiol and magnolol. These factors were the type of explants and the combination and concentration of plant-growth regulators. Green, compact, nodular organogenic callus was obtained from leaf explants in a medium fortified with Murashige and Skoog salts and supplemented with 1.5 mg/L 2,4-dicholorophenoxyacetic acid and 1.5 mg/L kinetin. Shoots multiplication from callus cultures was achieved in the Murashige and Skoog (MS) medium with 1.5 mg/L thidiazuron (TDZ). Phenol secretion was controlled by the addition of 250 mg/L of activated charcoal. For rooting, shoots were transferred to MS medium supplemented with several auxins. After root induction, the plants were hardened in earthen pots containing sand, soil, and vermiculite. The contents of honokiol (HK) and magnolol (MG) were determined in different plant materials by high-performance liquid chromatography-diode-array detection techniques. This analysis revealed that the honokiol and magnolol content in aerial and underground parts of micropropagated M. dealbata were higher than that observed in wild plants (both 6 months old). Our results suggest that conservation of M. dealbata is possible by means of in vitro multiplication of leaf-derived callus. The usefulness of M. dealbata regeneration and production of HK and MG may be attributed to the proper selection of explant sourcing and identification of the correct growth medium to support adequate growth. This careful selection of explants and growth medium leads to a very useful source of plant material for pharmacological and phytomedicinal screening applications and, above all, would safeguard this plant species from the threat of extinction.

Production of honokiol and magnolol in suspension cultures of Magnolia dealbata Zucc.

Honokiol and magnolol, important anxiolytic and anti-cancer agents, have been produced in cell-suspension cultures of the endangered Mexican plant Magnolia dealbata Zucc. In vitro cultures of the plant were established, and the accumulation of honokiol and magnolol in callus and cell-suspension cultures was measured. Leaf samples were the best explants for callus establishment and metabolite production, and Murashige and Skoog medium supplemented with 1 mg/L 2, 4-dichlorophenoxyacetic acid and 1 mg/L kinetin yielded 2.3 mg/g of honokiol and 5.9 mg/g of magnolol. Bacterial and fungal contamination was inhibited with a multiple-step tissue sterilization procedure. Oxidation was inhibited with 1 g/L activated charcoal. Cell-suspension batch cultures derived from friable callus obtained from leaves of this species were grown for 30 days in shaker flasks containing Murashige and Skoog medium. Throughout the growth cycle, honokiol and magnolol levels, fresh and dry weight, and sucrose uptake were determined. The effects of carbon source concentration on biomass accumulation and the synthesis of bioactive compounds were studied. By using 3 mL of inocula supplemented with 3% (w/v) sucrose, maximum yields of honokiol (8.1 mg/g) and magnolol (13.4 mg/g) were obtained after 25 days. These yields were 300% and 382%, respectively, of the yields of honokiol and magnolol obtained from field-grown plants.

Morfogénesis in vitro de dioon merolae de Luca, Sabato & Vázquez-Torres (zamiaceae, cycadales) a partir de megagametofitos y embriones cigóticos / In vitro morphogenesis of dioon merolae de Luca, Sabato & Vázquez Torres (zamiaceae, cycadales) from megagametophytes and zygotic embryos / Morfogênese in vitro de dioon merolae de Luca, Sabato & Vázquez-Torres (zamiaceae, cycadales) a partir de megagametofitos e embriões zigóticos

A partir de explantes de embriones cigóticos y megagametofitos fueron inducidos cultivos organogénicos de Dioon merolae del estado de Chiapas (México), cícada en peligro de extinción. El medio de inducción Litz consistió en los macronutrientes del medio B5, los micronutrientes del medio MS y los compuestos orgánicos glutamina (400mg·l-1) arginina (100mg·l-1), asparagina (100mg·l-1), sacarosa (60g·l-1), gellan-gum (4g), y suplementado con 0; 0,45; 2,26; 4,52 y 9,05μM αcido 2,4-diclorofenoxiacético (2,4-D), y 0; 2,32; 4,60; 9,30 y 13,90μM kinetina (K), con un arreglo de 5´5 con un diseño de bloque seleccionado al azar. Los cultivos fueron mantenidos en oscuridad a 25°C y el callo fue subcultivado en medio fresco cada cuatro semanas. La iniciación del callo ocurrió en un amplio rango de combinaciones de reguladores de crecimiento en explantes de megagametofito. El callo en explantes de embriones cigóticos se formó en pocas combinaciones. La formación de brotes adventicios ocurrió solo en callos que provenían de combinaciones con K y 2,4-D. A través del análisis histológico de secciones longitudinales de embriones cigóticos se detectaron células meristemáticas apicales de brote y raíz, y en megagametofitos la formación de elementos de conducción similares a traqueidas y raíces coraloides. Esta técnica presenta gran potencial para la preservación de especies de cícadas en peligro de extinción.

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Chiapas State (México) endangered cycad species, Dioon merolae. The Litz induction medium consisted of B5 major salts, MS medium minor salts and the organics glutamine (400mg·l-1), arginine (100mg·l-1), asparagines (100mg·l-1), sucrose (60g·l-1), gellan gum (4g), and supplemented with 0, 0.45, 2.26, 4.52 and 9.05μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0, 2.32, 4.60, 9.30, 13.90μM kinetin (K), arranged as a 5´5 factorial in a randomized block design. Cultures were maintained in darkness at 25°C, and callus was subcultured onto fresh medium at 4 week intervals. Callus initiation occurred on a wide range of plant growth regulators (PGR) combinations from megagametophyte explants. In comparison, callus initiation from explanted zygotic embryos occurred on few PGR combinations. Adventitious shoot induction occurred from callus on formulations with K and 2,4-D. Through the histological analysis of longitudinal sections of zygotic embryos were detected apical meristematic cells of the shoot and root and in megagametophytes the formation of elements similar to tracheids and coralloid roots. This technique has a great potential for preservation of the highly endangered cycads.

A partir de explantes de embriões zigóticos e megagametofitos foram induzidos cultivos organogênicos de Dioon merolae do estado de Chiapas (México), Cicas em perigo de extinção. O meio de indução Litz consistiu nos macronutrientes do meio B5, os micronutrientes do meio MS e os compostos orgânicos glutamina (400mg·l-1) arginina (100mg·l-1), asparagina (100mg·l-1), sacarose (60g·l-1), goma gelana (4g), e suplementado com 0; 0,45; 2,26; 4,52 e 9,05μM αcido 2,4-diclorofenoxiacético (2,4-D), e 0; 2,32; 4,60; 9,30 e 13,90μM kinetina (K), com um arranjo de 5×5 com um desenho de bloco selecionado aleatoriamente. Os cultivos foram mantidos no escuro a 25°C e o calo foi subcultivado, em meio fresco, cada quatro semanas. A iniciação do calo ocorreu em uma ampla faixa de combinações de reguladores de crescimento em explantes de megagametofito. O calo em explantes de embriões zigóticos se formou em poucas combinações. A formação de brotos adventícios ocorreu somente em calos que provinham de combinações com K e 2,4-D. Através da análise histológica de secções longitudinais de embriões zigóticos se detectaram células meristemáticas apicais de broto e raiz, e em megagametófitos a formação de elementos de condução similares a traqueídes e raízes coralóides. Esta técnica apresenta grande potencial para a preservação de espécies de Cicas em perigo de extinção.